Method and apparatus for electrophoretic focusing

ABSTRACT

An apparatus and method is provided for obtaining a preparative-scale, free-fluid electrophoretic separator with high resolution as well as an analytical capability commensurate with capillary zone electrophoresis. The electrophoretic focusing apparatus and method of the present invention combines features of electrophoresis and isoelectric focusing to accomplish large scale purifications and fractionations that have not previously been possible, and features a separation chamber bounded by precision-pore, insulated screens, a plurality of purge chambers, a plurality of electrode chambers, and a plurality of pump means. The separation device of the invention is capable of high speed of separation and short residency of sample through the use of high voltage gradients which are produced by relatively low voltages applied across the narrow chamber dimensions. The present invention is also highly flexible, with operation in either a constant electric field, continuous flow mode or in a linearly varying electric field, batch mode, and both modes permit scanning of the sample fraction content and display in a conventional histogram format. The present apparatus and method thus achieves high resolution of separation in an analytical or a preparative mode through a practically unlimited scale-up potential, and controls the adverse effects of Joule heating and electrohydrodynamics on the electrophoretic separation procedure.

This application is a divisional application of U.S. application Ser.No. 09/277,944 filed Mar. 29, 1999 now U.S. Pat. No. 6,171,466.

FIELD OF THE INVENTION

The invention relates in general to an apparatus and method forachieving electrophoretic focusing, and in particular to an apparatusfor achieving electrophoretic separation and purification which ischaracterized by a separation chamber formed between two precision-pore,insulated screens and which also includes inlet and outlet ports, aplurality of purge chambers for extracting extraneous fractions and forproviding thermal cooling, a plurality of electrode chambers to providea transverse electric field in the separation chamber, and pumping meanssample, carrier buffer and electrode rinse buffer through the apparatus,and a method of employing this apparatus to achieve separation andcollection of a desired component from a biological or chemical sample.

BACKGROUND OF THE INVENTION

There are two electrokinetic methods that have had success separatingbiological materials, namely, zone electrophoresis and isoelectricfocusing. Electrophoresis is the movement of suspended or dissolvedcharged particles in response to an applied electric field. The rate ofmotion depends upon the charge, size and shape of the particles andspecific properties of the solvent buffer and its container. In zoneelectrophoresis, the components in a short sample zone are separated bythe action of the electric field. The injection of a narrow, uniformzone and the absence of dispersive fluid flows are necessary conditionsfor successful operation. Significant sources of dispersion are: 1)uneven (parabolic) flows; 2) electrohydrodynamic flows; 3) moleculardiffusion; 4) thermal convection; 5) sedimentation; 6) thermally inducedsample mobility variations; and 7) electroosmosis.

In continuous zone electrophoresis (CFE), the electrolyte solution flowsin a direction perpendicular to the electric field and the mixture to beseparated is inserted continuously into the flowing solution. Componentsof the mixture are deflected according to their electrophoreticmobilities and can be collected continuously after their migration.Svensson and Brattsten were the first to report a method for carryingout electrophoresis continuously. They used a lateral electric field ina narrow plexiglas box packed with glass powder as an anti-convectivemedium. Durrum modified the above configuration by replacing theglass-filled box with a filter paper curtain, hanging in a free vaporspace. While both of these methods demonstrated continuouselectrophoresis, they both used a stabilizing medium. Anti-convectivemedia cause many problems such as reduction of the flow capacity bytheir presence, electroosmosis in the interstices, adsorption of thesample and “packing or eddy diffusion”. Efforts were then made to docontinuous electrophoresis in a free fluid. Bier in 1957 reported thefirst continuous flow electrophoresis device which could separate twoprotein solutions by adjusting the buffer pH relative to the isoelectricpoint of one of the solutions. The device which he described as“continuous free-boundary flow electrophoresis” did not take place in asingle rectangular chamber and did not produce a separation of highpurity. Dobry and Finn (U.S. Pat. No. 3,149,060) were the first toreport continuous flow free fluid electrophoresis in a rectangularchamber with a cross-section of low aspect ratio, hence providing littleresistance to thermal convective flow disturbances. This configurationwas limited to very low electric fields and required the use of bufferthickening agents to suppress convective eddies. Philpot described acontinuous flow electrophoresis system with the electric field appliedacross (perpendicular to) a thin film of liquid. He later wrapped histhin film geometry into a thin annulus surrounded by two concentriccylinders (electrodes). The outer cylinder rotated to provide astabilizing velocity gradient.

Although a large throughput, 10 g/hr, was accomplished by the Biostream,its resolution was poor. This was followed by forced flowelectrophoresis devised by Bier for the large scale purification of asingle component in a mixture. Giddings extended this development withfield flow fractionation wherein an electric field has been just oneexample of the force field deflecting the sample across the narrowplane. The need for flat, uniform surfaces that also serve to isolatethe electrode arrays have slowed this development. Mel in 1959 reportedthe first use of a high aspect ratio rectangular separation chamberusing a lateral electric field. The “thin” chamber of 0.7 cm thicknessprovided the necessary wall interaction to suppress thermal convectiveflows to the extent that a less viscous free flow buffer could be used.This design served as the impetus for the development of theconventional CFE machines of the 60's and 70's with their chambercross-section of high aspect ratio and laterally directed electricfields. During this time frame, Hannig and his co-workers developed CFEby making the chamber cross-sections even thinner, approaching 0.25 cmfor some designs. Unfortunately, the gains made in suppressing thermalconvection were wiped out by electrohydrodynamic interaction withintrinsic chamber fluid flows to cause crescent-shaped distortions.Nevertheless, a variety of CFE instruments were manufactured accordingto the designs of Hannig (in Germany) and Strickler (in the US) (U.S.Pat. No. 3,412,008) and several hundred instruments were used inlaboratories around the world. Rhodes and Snyder subsequently devised atechnique to minimize these flow distortions (U.S. Pat. No. 4,752,372).

The concept of counterflow to oppose the electrophoretic migration wasfirst described to the inventors by Griffin and McCreight as a means toattenuate the crescent shaped distortion in CFE chambers. Richmansubsequently patented a similar counter-flow method where axial bands ofelectroosmotic coatings of varying zeta potential would “straighten”distorted sample bands (U.S. Pat. No. 4,309,268). The method wasimpractical because most coatings change with time and there exists nospectrum of coatings with respect to zeta potential. A more practicalapproach that did not use counter-flow was suggested by Stricklerwherein the CFE was divided into two vertical compartments, each with adifferent wall coating, so that the combined electroosmotic flow wouldyield a more coherent sample band. Subsequently, Ivory used counter-flowto increase sample residence time in a recycling CFE. Egen, et al. havealso devised a counter-flow gradient focusing method (U.S. Pat. No.5,336,387).

While the crescent phenomenon was long known to cause untenable samplestream distortion in CFE instruments, it was not until 1989 that Rhodesand Snyder showed that electrohydrodynamics transforms initiallycircular sample streams into ribbons that initiate the crescent shapeddistortions. The operation of CFE devices was labor intensive andunreliable due to contamination of the closely spaced chamber walls andthe resultant electroosmotic flow variations through the chamber.

Isoelectric focusing (IEF) is an electrophoretic technique that adds apH gradient to the buffer solution and together with the electric fieldfocuses most biological materials that are amphoteric. Amphotericbiomaterials such as proteins, peptides, nucleic acids, viruses, andsome living cells are positively charged in acidic media and negativelycharged in basic media. During IEF, these materials migrate in thepre-established pH gradient to their isoelectric point where they haveno net charge and form stable, narrow zones. Isoelectric focusing yieldssuch high resolution bands because any amphoteric biomaterial whichmoves away from its isoelectric point due to diffusion or fluid movementwill be returned by the combined action of the pH gradient and electricfield. The focusing process thus purifies and concentrates sample intobands that are relatively stable. This is a powerful concept that hasyielded some of the highest resolution separations, especially whencoupled with electrophoresis in two-dimensional gels. Unfortunatelythere are drawbacks to IEF that have limited its applications. The rateof electrophoretic migration of each charged species decreasesprogressively as it approaches its isoelectric point and long residencetimes are required for high resolution. Proteins have reduced solubilityat their isoelectric point although precipitation of the concentratedbands can be minimized by addition of detergent. Additional problemsrelate to the commercial amphoteric solutions, including: 1) difficultyof extracting the separated proteins, peptides, etc., from theamphoteric solutions because of their similar physical properties andinteractions; 2) chemical toxicity; 3) handling problems; and 4) cost.

IEF had its practical beginning in the mid-1950's when Kolin firstdemonstrated the concept of focusing ions in a pH gradient by placing amolecular sample between an acidic and a basic buffer and applying anelectric field. Although the constituents focused rapidly, the gradientsoon deteriorated due to the concurrent electrophoretic migration of allof the buffering ions. The synthesis of stable carrier ampholytes byVesterberg and their successful commercial development led to broad usein gels or other restrictive media to suppress electroosmosis andthermal convection during analytical separations.

The high resolution achieved by IEF encouraged many attempts to developa preparative version of the process. This proved to be much moredifficult for IEF than zone electrophoresis because of the variablefluid properties and sample characteristics within the chamber leadingto changing values of electroosmosis and thermal convection during theseparation. Various CFE devices were modified to run with an amphotericmixture instead of buffer but the problems (long focusing time requiringa slow flow through the chamber, pH drift toward the cathode, reducedvoltage/current levels for acceptable heating and convection) becameinsurmountable. A. J. P. Martin described a means of performinglarge-scale isoelectric focusing by connecting a number of separationchamber in series via membranes. By circulating the fluids in eachcompartment through external coolers, Martin claimed that the removal ofheat had been solved. Since the only pH shift occurred across themembranes, the pH gradient was quite steep between chambers. Bierfurther developed the external cooling system, added sensors anddemonstrated the improved focusing with recycling (U.S. Pat. No.4,362,612). Bier added a stabilizing assembly rotation to the membranesegmentation and a novel collection system (U.S. Pat No. 4,588,492)which led to the Roto-Phor from Bio-Rad (Hercules, Calif.). Righetti hasalso extended the multi-compartment concept by using membranes, cast andpolymerized with the desired amphoteric molecules inside, to establishthe pH gradient rather than preparing a constant pH in each compartment.The Iso-Prime system (Hoefer Instruments, San Francisco, Calif.) isbased upon a stack of membranes with buffer between them. The pHgradient develops rapidly and the proteins move through the membranesuntil they reach the cell with the pH equal to their isoelectric point.Although the membranes stabilize the focusing process, they becomeclogged if the protein precipitates in them.

Thus, prior methods of isoelectric focusing have suffered from the manydrawbacks outlined above, and have also been hindered by problems duringthe transition from an analytical system to a preparative system thathave limited its intended use. It is thus highly desirable to develop afocusing system for separating biological molecules and other componentsin a mixture which is able to avoid all of the problems of the prior artand which can achieve high resolution of separation in an analytical ora preparative mode through a practically unlimited scale-up potential.It is also highly desirable to develop an electrophoretic focusingsystem which can control the adverse effects of Joule heating andelectrohydrodynamics on the electrophoretic separation procedure.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a preparative-scalefree-fluid electrophoretic separator with high resolution as well as ananalytical capability commensurate with capillary zone electrophoresis.The particular mode of high-resolution separation as provided by thepresent invention, which is referred to as electrophoretic focusing,combines features of electrophoresis and isoelectric focusing toaccomplish large scale purifications and fractionations that have notbeen possible before now.

It is another object of the present invention to develop a separationdevice capable of high speed and short residency through the use of highvoltage gradients. These high voltage gradients are produced byrelatively low voltages applied across the narrow chamber dimensions.Another object is flexibility with operation in either a constantelectric field, continuous flow mode or in a linearly varying electricfield batch mode. Both modes permit scanning of the sample fractioncontent and display in a conventional histogram format. The goal of highresolution of separation can be achieved through the use of the presentinvention in an analytical or a preparative mode through a practicallyunlimited scale-up potential. A further goal is to control the adverseeffects of Joule heating and electrohydrodynamics.

These and other objects and benefits are achieved by the use of thepresent invention which provides a number of innovations and insightswith regard to fundamental fluid and thermal geometries and operations.The focusing is accomplished with a minimum of sample migration whichleads to a higher resolution in a shorter time. Adiabatic thermalconditions in the lateral (scale-up) dimension permit a large increasein throughput at no apparent loss of resolution. Active cooling limitsthe maximum chamber temperature and its relationship to the chamberorientation and buffer fluid transport is such as to limit thermalconvection. Porous, rigid screens permit a controlled focusingcross-flow which balances the electrophoretically-driven samplevelocity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a side schematic view of the separation chamber of the presentinvention, taken in the axial and transverse directions.

FIG. 2 is a side schematic view of the separation chamber of the presentinvention, taken in the lateral and transverse directions.

FIG. 3 is a schematic view of one of the injector/collectors of thepresent invention.

FIG. 4 is a side schematic view of an alternative embodiment of theseparation chamber of the present invention, taken in the axial andtransverse directions.

FIG. 5 is a side schematic view of an alternative embodiment of theseparation chamber of the present invention, taken in the lateral andtransverse directions.

FIG. 6 is a schematic view of an alternative embodiment of one of theinjector/collectors of the present invention.

FIG. 7 is a side schematic view of a modified form of the separationchamber of the present invention.

FIG. 8 is a graphic representation of the electric field lines of anumerical solution of equations relating to the electric fieldcomponents of the apparatus of the present invention.

FIG. 9 is a side schematic view of an alternative embodiment of theseparation chamber of the present invention, taken in the axial andtransverse directions.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, there is provided anelectrophoretic focusing apparatus and method which is useful inachieving the separation and purification of particular components of amixture of biological or chemical materials. The general purpose of theinvention is a continuous processing system that separates and purifiesany soluble or microparticulate sample that acquires a surface electriccharge when immersed in a polar (e.g. aqueous) fluid environment. Itcombines the best features of electrophoresis and isoelectric focusingin a novel device that incorporates a combination of transverse electricfield gradient and buffer flow field to focus and collect any selectedbiological component. Although the high resolution achievable byfocusing is familiar to isoelectric focusing, electrophoretic focusingavoids many of its problems, such as the need for complex buffers andthe long times required for the molecules to reach their isoelectricpoint. This new concept incorporates a large-gap chamber and control ofall sources of sample dispersion. The design of the electrophoreticfocusing chamber combined with the orientation and magnitude of theelectric fields and buffer flows are planned to eliminate sampledispersion. The large gap will keep sample away from the walls as wellas increase its throughput.

Many research and applications tasks with biological materials require alarge source of highly purified biologically active molecules. Thediverse supply of materials for biotechnology ranging from plants togenetically derived sources are placing increased demands on separationand purification. Existing preparative separation techniques yieldproducts with a variety of impurities that can be measured analyticallybut not removed. Analytical techniques have been perfected in recentyears but attempts to scale these techniques into larger production haverelied on generally increasing the physical dimensions instead ofinvestigating a new technique. It is an advantage of the focusing deviceof the present invention that it will be able to purify biologicalmaterials in amounts and to purity levels above those now obtainable.

The principle of electrophoretic focusing utilized in conjunction withthe present invention consists of opposing the electrophoretic samplevelocity with a uniform fluid flow transverse to the direction ofcarrier flow through the chamber. Basically, this is the concept ofcounterflow which is discussed above. The uniqueness of this inventionis how this principle is used in conjunction with both constant andvarying voltage fields to achieve a novel and powerful method ofcontinuous sample separation. This result is achieved by using acombination of electrode arrays and insulated screens to provide theelectric field gradient and uniform transverse flow necessary forfocusing.

If the electric field is configured in the transverse direction (insteadof the lateral direction as with CFE), electroosmotic flow becomesnegligible and the viscous parabolic flow is orthogonal to the migrationdirection and hence also ceases to be a factor. Since the transversemigration is now in the narrow chamber dimension, the sample residencetime is quite short and resolution will suffer. However, if a cross-flowis used, the sample will be held in the chamber by the cross-flow, thusimproving the resolution by some calculatable amount. This solution tothe problems of CFE has been considered by past inventors but theproblem of the area electrode/chamber broad wall has kept this idea fromrealization. As the details of the invention show, this problem issolved by a unique utilization of micro-pore, thin, rigid, insulatingscreens.

The electrophoretic separator of the present invention is primarilycharacterized by a separation chamber formed between two precision-pore,insulated screens. The perforations permit transverse fluid flow throughthe chamber to effect a separation of one or multiple species and alsoto provide cooling in the chamber interior. This unique use ofcross-flow focuses one sample fraction continuously in the chamber whenusing a constant electric field to oppose the cross-flow oralternatively focuses a mobility spectrum of the sample in the chamberwhen using a linearly varying electric field. Since the separation iscarried out in the direction transverse to the carrier buffer flow, thefocusing is accomplished with a minimum of sample migration which leadsto a higher resolution in a shorter time. The relatively shorttransverse dimension allows the use of a high voltage gradient derivedfrom a low source voltage. When using low voltage gradients (to 100V/cm), the sample is injected and collected in singular or multipleports so that the chamber is only partially filled with sample. Atvoltage gradients from 100 V/cm to 1000 V/cm, the chamber thickness isgreatly reduced so that the sample fills the entire chamber. Thisconfiguration provides a homogeneous medium between the chamber wallsand eliminates conductivity gradients which produce destructivecirculatory flows through Joule heating and electro-hydrodynamics.

Another problem with CFE is the method of sample collection. Theseparated fractions must be collected by a finite number of collectionports which ultimately limit resolution. Collection for a batch process,such as chromatography or capillary electrophoresis (CE), poses no suchproblem as each separate fraction can be individually collected over avariable time interval rather than a limited fixed distance intervalbetween each adjacent collection port as with CFE. This invention cancollect fractions as a function of time by varying the crossflowvelocity to produce a histogram similar to that obtained fromchromatography or capillary electrophoresis.

The present invention is capable of operation as a true focusing devicein the same manner as IEF except that no pH gradient is utilized. Alinear varying electric field gradient is produced by an electrode arraywith a parabolic voltage distribution impressed on the array. Thissystem uses a homogeneous conventional buffer system in contrast to thecomplex, multi-component systems needed for conventional IEF. Sinceelectrophoretic focusing can be done with either a constant voltagegradient or with a linearly varying voltage gradient, the two respectiveconfigurations of the present invention will be described separately.

I. Constant Voltage Gradient Configuration

FIGS. 1 and 2 show the total chamber (or simply, the chamber) and thedifferent flow regions. The chamber is comprised of a plurality of flowregions or sub-chambers, such as the five elements 1, 4, 5, 26, and 27shown in FIG. 1. In the preferred embodiment, the separation chamber 1is bounded by two fine mesh precision-pore, insulated screens, 2 and 3.Carrier buffer enters the separation chamber through the inlet manifold15 and port 21. The buffer flows through the chamber as shown with acenter plane velocity of U_(c) and exits the separation chamber via theexit port 24 and manifold 18. Sample is injected in the form of a laminathrough the injection port 40 in the injector 12 shown in detail in FIG.3. The injector is made of glass-coated metal tubing and located in thechamber as FIG. 2 shows. Note that the sample lamina does not fill theentire separation chamber width (y-direction). Separated sample iscollected through slit 40 in the collector 13. Sample can still beinjected as a lamina through an injection port 40 without requiring theinjector 12 by placing the injector port 40 in the center of theseparation chamber entrance wall. Carrier buffer will now enter theseparation chamber through flanking buffer entry ports 21 and associatedmanifolds 15 located in each edge of the entrance wall. The convergingbuffer flows will reduce the thickness of the sample lamina. In asimilar manner, separated sample can be collected through a collectionport 40 located in the center of the separation chamber exit wall withthe buffer exiting the chamber through two flanking ports 24 andmanifolds 18 located in each exit wall, as illustrated in FIG. 9.

As best shown in FIG. 1, adjacent co-directed Uptake place in the purgechambers 4 and 5. These flows enter through inlet manifolds 14, 16 andinlet ports 20, 22. The flows exit through the manifolds 17, 19 andports 23, 25. An electric field E_(o) is impressed in the chamber byelectrodes 10 and 11. These electrodes are located respectively inelectrode chambers 26 and 27. Rinse flows of center plane velocity U_(e)take place in these chambers through manifolds 28, 30 and ports 29, 31.Membranes 8, 9 isolate the electrode chambers to contain electrolysisgas and products which are swept away by the electrode rinse flow U_(c).The membranes 8, 9 are rigidized by the precision-pore, insulatedscreens 6, 7. This is accomplished by keeping the pressure in theelectrode chambers greater than that in the rest of the chamber. Thisallows electric current to flow through the membranes while keepingelectrolysis products and flow disturbances confined to the electrodechambers. A focusing flow velocity V_(o) is established in theseparation chamber 1 by a fluid flow which enters through inlet ports 35and manifolds 37 located in the purge chamber 5. This flow exits thepurge chamber 4 through exit ports 34 and manifolds 36. This flow V_(o)is rendered uniform in the separation chamber by virtue of the smallpores in screens 2 and 3 (e.g., roughly 0.006 inch diameter) and thelarge purge chamber volume which serves as a flow manifold. Twoevacuated (38) glass walls 39 bound the chamber in the lateral dimensionas FIG. 2 shows.

It is important to eliminate lateral temperature gradients in theseparation chamber 1. The laterally directed electrode rinse flows U_(e)could possibly cause lateral temperature gradients to exist in theseparation chamber 1. For this reason, the rinse flows in the electrodechambers are oppositely directed as FIG. 2 shows. To further attenuatethese lateral temperature gradients, baffles can be placed laterally inthe electrode chambers and spaced in the axial direction. Flows inalternating directions can then be impressed by an appropriate manifoldsystem. These lateral temperature gradients originating in the electrodechambers are further averaged out by the purge flow Up taking place inthe purge chambers 4 and 5.

If the upper electrode 10 is negative and the bottom electrode 11 ispositive, an electric field E_(o) exists in the separation chamber 1which will cause a negatively charged sample to migrate down (transversedirection) under the influence of the electric field E_(o) against theuniform transverse focusing flow velocity V_(o). Consider a samplefraction of electrophoretic mobility, μ_(i)=V_(o)/E_(o) that has beeninjected through the port 12 located on the separation chamber centerplane. The sample fraction μ_(i) will remain at the center plane of theseparation chamber 1 and move through it with a carrier buffer velocityV_(c) and be collected at the exit port 13. All other sample (mobilitydifferent than μ_(i)) will exit either through port 24 in the separationchamber or through ports 23 and 25 in the purge chambers. A samplefraction scan can be made by varying V_(o). The effluent from collectionport 13 enters an ultraviolet detector and is displayed as aconventional histogram.

Thus, by varying the transverse focusing flow U_(o) against a constantelectric field E_(o), a scan of the fraction content of a sample can bemade. This type of scan of a sample is unique in a separation devicesince the peak histogram is a function of the time rate of change of thefocusing velocity V_(o) and is given by μ_(i)=V_(o)/E_(o). The time rateof change V_(o) is controlled by a precision computer controlled pump.This allows real time control of the separation process. Continuoussample collection can be made by stopping the scan at a peak ofinterest, or made after the complete scan has been made by recoveringthe transverse velocity V_(o) corresponding to a peak of interest.

The peak values are detected by a liquid chromatography flow cell anddetector system and fed back into the computer to achieve a feed-backcontrol system. Cooling of the electrode chambers 26, 27 is provided bythe electrode rinse flow while the purge flow Up provides cooling forthe rest of the chamber. The flow velocity U_(p) in the purge chambers4, 5 may be up to ten times that in the separation chamber 1 in order toaccomplish this purpose. The pore size of the screens 2, 3 is small(presently 0.006 inch hole, 34% open area) and thickness 20 gauge. Whilethe small holes will dampen disturbance flows between the separationchamber 1 and the purge chambers 4, 5, it is advisable to considerpressure drops in the separation and purge chambers so that b_(p)²/b_(c) ²=U_(p)/U_(c) where b_(p) and b_(c) are the thicknesses of thepurge and separation chambers respectively. The port 40 shown in FIG. 3confines the sample stream to the center region of the chamber. Thisconfiguration avoids the adverse effects of electroosmotic flows at theend walls. The evacuated glass side walls 39 preclude heat transfer inthe lateral direction as FIG. 2 shows. This condition eliminates anyvariance in this direction so that scale-up of the sample stream widthis unlimited.

Referring to FIG. 2, the focusing flow V_(o) causes a temperaturegradient in the transverse direction as it brings cooler flow from thepurge chamber 5 into the separation chamber 1. Also, referring to FIG.1, the purge flow which cools the separation chamber is heated as itmoves through the chamber giving rise to an axial temperature gradientin the separation chamber. The first gradient gives rise to a clockwisecirculation when the chamber is in the vertical orientation, while thesecond gradient gives rise to a similar clockwise circulation when thechamber is in the horizontal orientation. Mathematical models show thatthe circulations are attenuated to a much greater extent by the chamberwalls when the chamber is in the horizontal orientation. However, byutilizing internal injection and collection ports 12, 13 the effect ofthe circulations is eliminated since the disturbance flows occur in thefront of port 12 and behind port 13 and hence does not affect theseparation. Thus there is no effect of chamber orientation on thermalconvection disturbances with internal injection and collection ports.The above configuration is adaptable to voltage gradients up to about100 V/cm. However, if higher voltage gradients are preferred, somemodification should be considered.

The high voltage (second) configuration is characterized by a very thin(transverse thickness) separation chamber and the elimination of theinternal injection and collection ports 12 and 13. Sample is injectedthrough port 21 and collected through port 24. Hence, sample fills theentire separation chamber with no buffer zones which characterize thelow voltage configurations. The smaller heated volumes of the separationand purge chambers limit the chamber temperature at high electricfields. A significant advantage of having only sample in the separationchamber is the homogeneity of the electrical conductivity in the fielddirection. Since Joule heating and electrohydrodynamics both vary as theelectric field squared, and since the adverse effects of both aredependent on electric field gradients, it is important to eliminatethese gradients if high voltage gradients are to be successfully used.The high voltage configuration must be operated in the horizontalorientation. While lateral gradients are controlled by the insulatedends 39, transverse temperature gradients can be significant at highvoltage gradients and are exacerbated by the cool focusing cross-flowfrom purge chamber 5 into the separation chamber. The focusing flowV_(o) produces a gradient of increasing temperature from purge chamber 5through the separation chamber to purge chamber 4. This gradient cangive rise to significant circulation if the chamber is operated in thevertical orientation aligned with gravity. If the chamber is operated inthe horizontal orientation, this circulation is suppressed as ourmathematical models of the chamber configuration have shown. Since thechamber is cooled by the purge flows of velocity U_(p) emitting from thepurge manifolds, an axial temperature gradient is also developed in thechamber, however, it is generally much smaller than that produced by thetransverse gradient and is similarly suppressed by horizontal operation.

II. Varying Voltage Gradient Configuration

The third configuration shown in FIGS. 4, 5 and 6 can be described as atrue focusing system. The significant features of this innovation arethe use of a constant transverse flow and a varying transverse electricfield that is linear in the transverse coordinate. The electric fieldcan be described by:

Ez=B−Cz,

where z is perpendicular to the direction of the carrier buffer flow, Bis the field at z=0 and C is a linear constant. The correspondingelectrophoretic velocity of sample fraction i is:

v _(e)=μ_(i) E _(z)=μ_(i)(B−Cz)

Focusing is achieved by the imposition of a constant fluid flow of speedV_(o) with a direction opposing v_(e). The velocity of a single ionizedmolecule of the i^(th) sample fraction is:

dz _(e) /dt=−V _(o)+μ_(i)(B−Cz)  (1)

where z_(e) is the electrophoretic displacement of the fraction in thez-direction. Each fraction approaches a null position Z_(i) given by:

V _(o)=μ_(i)(B−Cz _(i))  (2)

Solving equation 1,

z _(e)=(z _(o) −z _(i))exp(−μ_(i) Ct)+z _(i)  (3)

where z_(o) is the initial ion position. For large t, the first termapproaches zero so that the final (focused) position z_(i) isindependent of the initial position, z_(o), and is the position obtainedfrom equation 2. The advantage of focusing is that each fraction movestoward a fixed position in the separation chamber. This is in contrastto zonal techniques where fractions move to relative positions withrespect to other fractions.

FIGS. 4 and 5 show a multi-chamber configuration similar to the firsttwo configurations. The separation chamber 1 is much wider in this casein order to accommodate the multitude of injection 12 and collectionports 13 necessary to handle the sample mobility spectrum of interest.The single electrodes of FIGS. 1 and 2 have been replaced with electrodearrays 10 and 11. Each element can be impressed with a specific voltageand is the mechanism by which the linear voltage gradient E_(Z) isproduced. For focusing, sample and buffer is injected through the portarray 12 with the separated fraction streams collected at the exit portarray 13. Flow occurs in the separation chamber 1 only between the portarrays 12, 13. Flow stagnation regions 42 and 43 respectively exist infront of port array 12 and in back of port array 13. The ports 21 and 24are only active initially during filling of the chamber. Thesestagnation regions serve both as electrical current and the fluid flowreturn paths. FIG. 7 shows a simplified schematic of the chamber with acoordinate system to facilitate a description of the electrode and flowfield needed for focusing and which will justify the port and electrodemodifications described above.

The total rectangular chamber is bounded by:

|x|<a; |y|<b and |z|<c, where y is the orthogonal axis to the x-z plane.The total chamber dimensions are double these values. It is separatedinto three segments by screens 2, 3 at |z|=e.

The linear electric field gradient E_(z) is provided by electrode arrays10 and 11 distributed along the line |z|=c and are kept at the followingvoltages:

 V=−{Ax+Bz+½C(x ² −z ²)}

where the constants A, B and C are independent and computer controlledto optimize the separation. The purpose of these electrodes is tomaintain this voltage V (which locally satisfies Laplace's equation,∇²V=0) throughout the separation chamber 1 and its four adjacentsegments 4, 5, 26, and 27. The insulating walls at |y|=b do not modifythis voltage distribution. The inflow and outflow ports 12, 13 at |x|=dwill not modify the electric field distribution provided each port iselectrically isolated and there are adequate gaps in the z-directionbetween the ports, as FIG. 6 shows. The corresponding electric fieldcomponents are:

E _(x) =A+Cx  (4)

E _(z) =B−Cz  (5)

Equation 5 produces focusing while equation 4 causes a linear variationin the axial ion motion affecting residence time which can be used topromote sample concentration.

To see how equations 4 and 5 satisfy ∇²V=0, a numerical solution is donefor the region bounded by |z|=c and |x|=a. The boundary condition at|z|=c is

V=−½C(x ² −z ²)

where A=0 and B=0 so that only the C terms are considered. The fieldlines for this solution are shown in FIG. 8. This computation was donefor a/c=20; note that the z-dimension is exaggerated in the display.Note that the field lines indicate that equations 4 and 5 are goodapproximations except near the walls at |x|=a and |z|=c. This region ofconformity is bounded by |z|=e and |x|=d and is the separation chamber.The stagnation regions 42, 43 also provide a return path for the currentconverging near the walls at |x|=a. These return paths assure that

E _(z) =B−Cz

in the separation chamber 1. In order to improve resolution, sample fromthe collection ports 13 can be recycled back into their correspondingentry ports 12 to form a recycling mode of operation. This mode will bea batch type for purposes of sample collection.

The preferred embodiment for detection and collection of the focusedsample streams is to use a UV scan in front of the collection ports 13to establish the position of the fractions and generate a conventionalhistogram for the fractions observed in the mobility spectrum currentlyunder observation. One port should be used to collect a fraction ofinterest by positioning that fraction over the collection port bymanipulation of either V_(o) or E_(o). This process is most easilyhandled through the use of computer control. Other fraction spectra canbe observed and/or collected by further manipulation of V_(o) or E_(o).

As one of ordinary skill in this art would recognize, the abovedescriptive embodiments are only exemplary of the present invention, andthere are numerous modifications and alternative modes that would fallwithin the scope of the invention, which is set forth in the claimsappended hereto.

What is claimed is:
 1. A method for separation and collection of atleast one component from a mixture of components comprising the stepsof: a. providing an apparatus comprising a separation chamber and aplurality of purge chambers, and establishing a first buffer flow in theseparation chamber in the axial direction, said first buffer flow havinga first flow rate; b. establishing a second buffer flow in each of atleast two of said purge chambers in the axial direction, said secondbuffer flow having a second flow rate, said second buffer fluid flowhaving a second flow rate higher than that of the first flow rate; c.establishing a third buffer flow in a plurality of purge chambersaxially adjacent to said separation chamber and separated from theseparation chamber by metal-coated glass screens; d. establishing afourth buffer flow into one of the purge chambers laterally through theseparation chamber, then into and out of a second purge chamber toprovide a uniform focusing fluid velocity in the separation chamber; e.introducing the mixture of components directly into the separationchamber flow entrance or through at least one injection port located inthe separation chamber interior; f. applying an electrical potentialtransversely across the separation chamber in the form of a constantvoltage gradient or a linearly varying voltage gradient to impartelectrophoretic velocity to the fractional components in the separationchamber in the transverse direction perpendicular to the first bufferflow direction and parallel to the third buffer flow direction; and g.withdrawing at least one separated sample component.
 2. A methodaccording to claim 1 wherein the separated sample is withdrawn at theflow exit of the separation chamber.
 3. A method according to claim 1wherein the separated sample is withdrawn through a single collectionport or from each of a plurality of collection ports.
 4. A methodaccording to claim 3 wherein the sample entering collection ports may berecycled back to the corresponding sample entry ports to form arecycling process which can improve resolution by increasing sampleresidence time.
 5. A method according to claim 1 wherein the sample isinjected with the carrier flow at the flow entrance of the separationchamber and is acted on by the combined influences of a constantelectric field and said third flow transversely across the separationchamber.
 6. A method according to claim 1 wherein one sample componentis maintained in the separation chamber while extraneous components arediscarded through the purge chambers.
 7. A method according to claim 1wherein the third buffer flow is adjusted to provide a transverselyvarying cross-flow velocity which allows any selected sample componentto be either analyzed or collected.
 8. A method according to claim 7wherein the selected sample component is collected at the flow exit ofthe separation chamber.
 9. A method according to claim 1 wherein thesample is injected through a single injection port into the separationchamber while carrier buffer enters the separation chamber at the fluidflow entrance of the separation chamber.
 10. A method according to claim9 wherein the sample is acted on by the combined influences of aconstant electric field and said third buffer flow transversely acrossthe separation chamber.
 11. A method according to claim 10 wherein onesample fraction is maintained in the separation chamber and arrives at asingle collection port in the separation chamber while extraneouscomponents are either discarded through the said purge chambers or flowaround the collection port and out of the separation chamber at thecarrier buffer flow exit.
 12. A method according to claim 1 wherein thesaid third buffer flow is adjusted to provide a transversely varyingcross-flow velocity which allows any selected sample component to beeither analyzed or collected at a single collection port.
 13. A methodaccording to claim 1 wherein the fraction spectrum of a sample may beanalyzed or collected by varying the flow of a pump in a linearvariation to present a time-dependent histogram.
 14. A method accordingto claim 1 wherein the sample and buffer solution is injected through aplurality of input ports located at the entrance region of theseparation chamber.
 15. A method according to claim 14 wherein thesample fractions are acted on by the combined influences of a linearlyvarying electric field and a third buffer flow transversely across theseparation chamber.
 16. A method according to claim 1 wherein the samplefractions migrate toward set transverse positions near the exit end ofthe separation chamber.
 17. A method according to claim 16 wherein thesample fractions may be scanned in the exit region of the separationchamber by a UV laser and detector system with sample fractions beingcollected in a single or multiple set of collection ports.
 18. A methodaccording to claim 17 wherein the sample fraction spectrum in theseparation chamber is fixed by the transverse chamber thickness with theremainder of the spectrum being diverted through screens into and out ofthe purge chambers.
 19. A method according to claim 18 wherein thesample fraction spectrum being viewed may be collected in singular ormultiple collection ports by varying the third flow rate of focusingfluid velocity.
 20. A method according to claim 16 wherein the samplefraction spectrum being viewed may be changed by varying the third flowrate of focusing fluid velocity.